Michael R. Deans - US grants

DESCRIPTION (provided by applicant): The senses of hearing and balance are mediated by the hair cells of the inner ear, sensory epithelial cells with specialized architectures optimized for the detection of movement. Hair cells detect motion via the mechanical deflection of a kinocilium and stereocilia bundle located at one edge of the apical cell surface. Only movements of the bundle towards the kinocilium generate an excitatory response. Consequently the proper polarization of the stereocilia bundle and coordination of bundle polarity between adjacent cells is necessary for accurate vestibular function. Auditory hair cells within the organ of Corti detect sound in a similar fashion and the bundle polarity of adjacent cells is also coordinated. This type of organization is called planar cell polarity (PCP) and has been described in a number of different tissues and species. Moreover, hair cells within the vestibular maculae are further organized into two groups with opposite bundle polarities patterned about an abrupt line of polarity reversal (LPR). This increases the range of detectable motion and sensitivity to motion in a single direction. However, despite the likely importance of stereocilia bundle polarity and hair cell patterning for vestibular function it is not known how bundle polarization is initiated and coordinated within the sensory epithelia. Within this research proposal the cellular mechanisms guiding the polarization of the stereocilia bundle and orientation of hair cells will be evaluated using a combination of transgenic, mutant and knockout mice. [The function of the essential polarity gene van gogh-like2 (vangl2) will be determined by analyzing hair cell PCP using a novel vangl2 knockout mouse (vangl2TMS). These results will be compared to the vangl2 mutant line looptail which has become a reference for inner ear PCP mutant analysis despite having semi-dominant phenotypic characteristics.] Next the cellular mechanism of Vangl2 function in coordinating bundle polarity between adjacent cells will be tested through the production and analysis of a vangl2 conditional knockout line (vangl2floxedATG) in which the polarity gene is deleted in a cell-specific manner. These experiments will determine whether polarity cues are propagated from cell to cell and whether vangl2 is necessary for initiating bundle polarity or maintaining bundle polarity during inner ear morphogenesis. In addition, other vangl2 mutations result in embryonic lethality and this will be avoided be generating ear-specific vangl2floxedATG conditional knockouts. These mice will enable a series of behavioral experiments to examine the effects of hair cell misorientation on vestibular and auditory function. PUBLIC HEALTH RELEVANCE: Project Narrative The proper morphogenesis and organization of specialized inner ear sensory receptors called hair cells is necessary for hearing and balance, and the loss of these cells is the primary basis of age-related deafness and balance disorders. This project is designed to determine how hair cells are oriented and patterned within the within the vestibular maculae of the utricle and saccule. Understanding these events should reveal the basis of some forms of vestibular dysfunction and will define critical parameters that must be met by therapeutic approaches that rely upon introducing replacement hair cells into the mature inner ear.

Project Summary During the assembly of neural circuits, newly born neurons migrate to specific locations and extend processes in stereotyped manners to contact the appropriate synaptic partners. This is most evident in the vertebrate retina which is organized into three nuclear layers separated by two intervening synaptic plexiform layers. Despite the fundamental nature of this organization little is known about the cellular mechanisms that position neuronal cell types and direct dendrite extension. This study examines the function of the atypical cadherin protein Fat3 which coordinates these events during amacrine cell development. Fat3 is an unusually large cadherin molecule with a mass of ~500Kd that contains 34 extracellular cadherin domains and is present throughout the developing inner plexiform layer (IPL). Analysis of knockout mice reveals that fat3 is necessary for directing the polarized extension of amacrine cell dendrites into the IPL as well as properly distributing amacrine cells between the inner nuclear layer (INL) and the ganglion cell layer (GCL). In the absence of fat3 ectopic amacrine cell dendrites elaborate outside of the IPL resulting in the formation of two additional synaptic layers. In this project the molecular and cellular mechanism(s) of Fat3 function will be determined using novel lines of fat3 knockout and conditional knockout mice. Specifically transgenic reporters will be used to identify morphological classes of amacrine and ganglion cells expressing Fat3 and distinguish between a general function for amacrine cell development versus a function in the assembly of specific neuronal circuits. Additional in vitro experiments will dissect the regulatory mechanisms that control Fat3 signaling including alternative splicing and dynamic interactions with different cytoplasmic proteins. Finally ganglion cell development will be analyzed to determine if Fat3 is also required for the morphogenesis and distribution of this cell type, and central projections will be examined to determine if Fat3 is necessary for axonal development. Although focused on the function of Fat3 in the retina, the proposed work will nevertheless provide important insight into the role of Fat cadherins during neurodevelopment and in other systems where they are highly expressed such as the inner ear and kidney.

DESCRIPTION (provided by applicant): In the vestibular system of the inner ear, motion is detected via the mechanical deflection of a bundle of stereocilia located at the top of sensory receptor hair cells. The bundle is morphologically and physiologically polarized because only movements of the bundle towards a lone kinocilium positioned at one side of the apical cell surface are able to produce excitatory responses. Thus the range of motion that can be detected by an individual hair cell is determined by the polarized orientation of the stereocilia bundle. As a result, in order to respond to the broadest range of motions the utricle and saccule contain thousands of vestibular hair cells arranged in radiating arrays spanning a range of nearly 360ps of stereocilia bundle orientations. This is achieved in part by dividing the hair cell between two groups divided by a Line of Polarity Reversal (LPR) that have opposing stereocilia bundle orientations and respond to motions in opposite directions. Our goal is to identify the genetic mechanisms that direct the development of planar polarity. This will be addressed through the course of the project using combinations of knockout and transgenic mouse models. Specifically we will test the hypothesis that the core Planar Cell Polarity (PCP) proteins establis an underlying ground polarity that coordinates the orientation of adjacent hair cells regardless of their position relative to the LPR, and that a second patterning mechanism positions the LPR. For these experiments the significance of the PCP-based ground polarity will be established using a combination of single and double knockouts mouse lines that prevent core PCP signaling. This will complement a genetic dissection of Wnt-signaling and its parallel role in positioning the LPR. Finally, additional factors directing formation of the LPR will be identified through genetic labeling and FACS-based isolation of hair cells with opposite bundle orientations followed by microarray analysis. Although focused on the development of vestibular planar polarity, we anticipate that this research will impact our understanding of auditory planar polarity as well as other organ systems that rely upon cellular polarization for growth or function

The cochlea is innervated by the bipolar sensory neurons of the spiral ganglia that relay sound information from sensory receptor hair cells to central auditory targets. Deafness due to acoustic trauma is associated with pathologies in both spiral ganglion neurons and the hair cells which they innervate and an important aspect of repairing the deafened cochlea is coaxing spiral ganglion neurons to re-innervate their hair cell partners. It is generally anticipated that hair cell re-innervation will involve similar cellular and molecular mechanisms to those guiding nascent hair cell innervation. Therefore, understanding all aspects of spiral ganglion development and hair cell innervation are important prerequisites of regeneration-based therapeutic strategies. A subset of neurons in the spiral ganglion is dedicated to a fundamentally important feedback circuit that provides neuroprotection in extreme noise and facilitates hearing and speech discrimination in background noise. This circuit is dependent on the Type2 spiral ganglion neurons (SGN2) that innervate the outer hair cells. The morphological development of SGN2s is unique because their peripheral axon projects beyond the inner hair cells before making a distinct 90° turn towards the base of the cochlea in order to synapse with 8 to 10 outer hair cells. While many aspects of SGN2 development and outer hair cell innervation are not known, our laboratory has found evidence that the planar cell polarity protein Vangl2 contributes to at least one step in this process; the turning event that directs the SGN2 peripheral axon to the base of the cochlea. The goal of this Exploratory/Developmental Research grant is to establish two basic properties of Vangl2 function during SGN2 peripheral axon turning with the expectation that this will form the foundation of a larger, independent line of research addressing spiral ganglion development. The first is to distinguish between autonomous and non-cell autonomous sites of Vangl2 function in the peripheral axon growth cone or organ of Corti. This will be accomplished using a vangl2 conditional knockout line previously generated by the lab in combination with Cre lines selected to spatially restrict vangl2 gene deletion. The second is to assay the relative contribution of two alternative non-canonical Wnt receptors and signaling pathways that have been demonstrated to function upstream of Vangl2 in other contexts. This will be established through genetic interaction assays based upon the hypothesis that if Vangl2 and an upstream receptor function in the same pathway, then removing both will enhance SGN2 turning phenotypes. While these experiments are focused on developmental processes guiding axon pathfinding and target cell innervation we anticipate that these events must be recapitulated during hair cell re-innervation and repair, and therefore the proposed research will advance therapies for repairing the deafened cochlea.

Project Summary The cochlea is innervated by spiral ganglion neurons, which relay sound information from sensory hair cells to central auditory targets. Deafness due to acoustic trauma is associated with pathologies in both spiral ganglion neurons and the hair cells which they innervate, and an important aspect of repairing the deafened cochlea is coaxing spiral ganglion neurons to re-innervate their hair cell partners. It is generally anticipated that hair cell re- innervation will require the reactivation of developmental mechanisms. Therefore, understanding early developmental events is an important prerequisite for regeneration-based therapeutic strategies. A subset of spiral ganglion neurons has nociceptive characteristics and are thus equipped to detect acoustic trauma, which may be important for preserving function. These are the type II spiral ganglion neurons, which constitute a minority of cochlear afferents but innervate all outer hair cells. The development of type II neurons is unique and facilitates outer hair cell innervation because their peripheral axons project beyond the inner hair cells. An important component of cochlear innervation is how the type II spiral ganglion neurons subsequently make a distinct 90° turn towards the cochlear base to synapse with multiple outer hair cells. While many aspects of outer hair cell innervation are unknown, our laboratories have found that two signaling pathways, planar cell polarity (PCP) signaling and Eph/Ephrin signaling, are required for the 90° turn that directs the peripheral axon towards the cochlear base. A similar phenotype occurs with loss of the transcription factor Prox1 suggesting that a regulatory hierarchy controls cochlear innervation. The goal of this research is to establish the relationship between these two signaling pathways by examining each in detail and relative to each other. This includes experiments in Aim 1 to distinguish between alternative mechanisms in which PCP proteins pattern the organ of Corti prior to innervation or signal directly to the growth cone. Since the Ephrin receptor EphA7 is also required for axon turning, in Aim 2 we will determine if these pathways are linearly organized or if they are parallel and redundant signals with each promoting turning. Remarkably the EphA7 promoter contains putative Prox1 binding sites suggesting that these guidance mechanisms may be transcriptionally regulated. This hypothesis will be tested further in Aim 3. While these experiments are focused on developmental processes, we anticipate that these are events which must be reenacted during hair cell re-innervation and repair, and therefore the proposed research will advance therapies for repairing the deafened cochlea.